GSM6589687: P60 bio rep 1; Mus musculus; RNA-Seq
A laparotomy was performed, and the ileum was removed and lavaged with PBS containing penicillin-streptomycin (PS; Invitrogen), then cut into 1-cm-long segments and placed over a sterile plastic rod. A super-ficial longitudinal incision was made along the serosal surface and the LM-MP was peeled off from the underlying tissue using a wet sterile cotton swab (Kulkarni et al., 2017) and placed in Opti-MEM medium (Invitrogen) containing Pen-Strep (Invitrogen). The tissues were then dissociated in Digestion Buffer containing 1 mg/ml Liberase (Sigma-Aldrich) in OptiMEM. Tissues from mouse 1 were dissociated in the Digestion buffer containing Liberase TH and tissues from mouse 2 were dissociated in the Digestion buffer containing Liberase TL. Dissociation was performed at 37°C for 30 minutes on a rotary shaker, after which the cells were centrifuged at 200g for 7 minutes, and the pellet was resuspended in ice cold ster-ile PBS. The cell suspension was passed through a 40 µm cell sieve and the resulting filtered cell suspension was again centrifuged at 200g for 7 minutes. This process of cell centrifugation and filtration was repeated two more times, after which the cells were resuspended in 1 ml ice cold sterile PBS. The repeated steps of serial cell washes and filtration removed clumps and debris and the viability of the resulting cell suspension was estimated to be >90% using Trypan Blue dye test. The cells were then processed through 10X Genomics Chromium V3.0 system according to the manufacturer's suggested workflow. The processing was done at the GRCF Core Facility at the Johns Hopkins University. 10x Genomics Chromium v3.0 3' gene expression assay